Ryanodine receptors (RyRs) are intracellular calcium channels that are particularly important in skeletal and cardiac muscle where they play a key role in excitation-contraction (E-C) coupling, the process by which neuron-induced depolarization of the plasma membrane (sarcolemma) causes release of calcium ions from the sarcoplasmic reticulum (via RyRs). Mutated and malfunctioning RyRs have been implicated in certain diseases of skeletal (malignant hyperthermia, central-core disease) and, quite recently, cardiac muscle (two forms of sudden cardiac death, heart failure). In cardiac muscle RyRs also play important roles in pacemaker activity and in the generation of arrhythmogenesis. By characterizing the structure of RyRs and their interactions with associated proteins we expect to advance our understanding of RyRs' roles in E-C coupling mechanisms and in heart and skeletal muscle diseases. Purified RyRs from either natural sources (mammalian skeletal & cardiac muscle) or from transfected cultured cells that express mutated receptors will be characterized by cryo-electron microscopy and single-particle image processing to generate three- dimensional reconstructions (3D-cryoEM) at 10-30 Angstrom resolution. The specific aims of this proposal are to: (1) Determine the location of surface-exposed amino acids and regulatory sites by tagging them with green fluorescent protein or other macromolecular ligands, and (2) Characterize dynamical aspects of RyR function at increasing resolution by 3D cryo-EM and time-resolved cryo-EM. Specifically, the mechanisms by which the numerous domains that comprise the structure interact with each other to regulate channel gating by means of very long-range (>100 Angstroms) communication will be determined. Additionally, a hypothesized disruption of interdomain interactions by mutations that cause skeletal and sudden cardiac death diseases will be characterized, as well as the structural transitions that occur upon RyR regulation by calmodulin.